The present invention relates to a method for spectral analysis of objects, i.e., spectral imaging. More particularly the present invention relates to an interferometer based method aimed at spectral imaging of a moving object, to determine the spectral intensity of each pixel thereof. Spectral intensity according to the invention may be due to light emitted (naturally occurring or induced fluorescence) from, reflected or scattered by and/or transmitted through the moving object, all are further referred hereinbelow as light emitted from the object.
Spectroscopy is a well known analytical tool which has been used for decades in science and industry to characterize materials and processes based on the spectral signatures of chemical constituents. The physical basis of spectroscopy is the interaction of light with matter. Traditionally, spectroscopy is the measurement of the light intensity emitted, transmitted, scattered or reflected from an object, as a function of wavelength, at high spectral resolution, but without any spatial information. Spectral imaging, on the other hand, is a combination of high resolution spectroscopy and high resolution imaging (i.e., spatial information).
A spectrometer is an apparatus designed to accept light, to separate (disperse) it into its component wavelengths, and measure the light's spectrum, that is the intensity of the light as a function of its wavelength. An imaging spectrometer, also referred herein as a spectral imager, is one which collects incident light from a scene and measures the spectra of each pixel (i.e., picture element) thereof.
Thus, spectral imaging is a technology which enables the measurement of the spectrum of light emitted from every point (pixel) of an object. A spectral imager is an instrument that measures and stores in memory for later retrieval and analysis the spectrum of light emitted from every point of the object which is placed in its field of view. A spectral image is a collection of spectra of the object measured by a spectral imager. It is usually organized as an intensity function defined in a three dimensional space in which two dimensions are of an image (x and y), and one is of a spectral axis (.lambda.). As such, a spectral image is usually referred to as a "cube" of data or "spectral cube".
There are three basic types of spectral imaging methods. These are (i) grating based spectral imaging, (ii) filters based spectral imaging (iii) and interferometer based spectral imaging.
In grating based spectral imaging systems, also known as slit-type imaging spectrometers, such as for example the DILOR system: [see, Valisa et al. (September 1995) presentation at the SPIE Conference European Medical Optics Week, BiOS Europe '95, Barcelona, Spain], only one axis of a CCD (charge coupled device) array detector (the spatial axis) provides real imagery data, while a second (spectral) axis is used for sampling the intensity of the light which is dispersed by the grating as function of wavelength. The system also has a slit in the first focal plane, limiting the field of view at any given time to a line of pixels. Therefore, a full image can only be obtained after scanning the grating or the incoming beam in a direction parallel to the spectral axis of the CCD in a method known in the literature as line scanning. The inability to visualize the two-dimensional image before the whole measurement is completed, makes it impossible to choose, prior to making the measurement, a desired region of interest from within the field of view and/or to optimize the system focus, exposure time, etc. Grating based spectral imagers are in use for remote sensing applications, because an airplane (or satellite) flying over the surface of the Earth provides the system with a natural line scanning mechanism.
It should be further noted that slit-type imaging spectrometers have a major disadvantage since most of the pixels of one frame are not measured at any given time, even though the fore-optics of the instrument actually collects incident light from all of them simultaneously. The result is that either a relatively large measurement time is required to obtain the necessary information with a given signal-to-noise ratio (sensitivity), or the signal-to-noise ratio is substantially reduced for a given measurement time. Furthermore, slit-type spectral imagers require line scanning to collect the necessary information for the whole scene. which may introduce inaccuracies to the results thus obtained.
Filters based spectral imaging methods can be further categorized into discrete filters and tunable filters. In these types of imaging spectrometers the spectral image is built by filtering the radiation for all the pixels of the scene simultaneously at a different wavelength at a time by inserting in succession narrow band filters in the optical path, or by electronically scanning the bands using acousto-optic tunable filters (AOTF) or liquid-crystal tunable filter (LCTF), see below. Similarly to the slit type imaging spectrometers equipped with a grating as described above, while using filter based spectral imaging methods, most of the radiation is rejected at any given time. In fact, the measurement of the whole image at a specific wavelength is possible because all the photons outside the instantaneous wavelength (band) being measured are rejected and do not reach the CCD.
Tunable filters, such as AOTFs and LCTFs have no moving parts and can be tuned to any particular wavelength in the spectral range of the device in which they are implemented. One advantage of using tunable filters for spectral imaging is their random wavelength access; i.e., the ability to measure the intensity of an image at a number of wavelengths, in any desired sequence without the use of a filter wheel. However, AOTFs and LCTFs have the disadvantages of (i) limited spectral range (typically, .lambda..sub.max =2.lambda..sub.min) while all other radiation that falls outside of this spectral range must be blocked, (ii) temperature sensitivity, (iii) poor transmission, (iv) polarization sensitivity, and (v) in the case of AOTFs an effect of shifting the image during wavelength scanning.
All these types of filter and tunable filter based systems have not been used successfully and extensively over the years for spectral imaging, because of their limitations in spectral resolution, low sensitivity, and lack of easy-to-use, non-sophisticated algorithms for interpreting and displaying the collected data.
The sensitivity advantage that interferometer based spectroscopy has over the filter and grating methods is known in the art as the multiplex or Fellgett advantage [see, Chamberlain (1979) The principles of interferometric spectroscopy, John Wiley and Sons, pp. 16-18 and p. 263].
A method and apparatus for spectral imaging which have advantages in the above respects are disclosed in U.S. Pat. No. 5,539,517, to Cabib et al., issued Jul. 23, 1996, which is incorporated by reference as if fully set forth herein, with the objective to provide a method and apparatus for spectral imaging which better utilizes all the information available from the collected incident light of the image to substantially decrease the required frame time and/or to substantially increase the signal-to-noise ratio, as compared to the conventional slit- or filter type imaging spectrometer.
According to this invention, there is provided a method of analyzing an optical image of a scene to determine the spectral intensity of each pixel thereof by collecting incident light from the scene; passing the light through an interferometer which outputs modulated light corresponding to a predetermined set of linear combinations of the spectral intensity of the light emitted from each pixel; focusing the light outputted from the interferometer on a detector array, scanning the optical path difference (OPD) generated in the interferometer for all pixels independently and simultaneously and processing the outputs of the detector array (the interferograms of all pixels separately), to determine the spectral intensity of each pixel thereof.
This method may be practiced by utilizing various types of interferometers wherein the OPD is varied to build the interferograms by moving the entire interferometer, an element within the interferometer, or the angle of incidence of the incoming radiation. In all of these cases, when the scanner completes one scan of the interferometer, the interferograms for all pixels of the scene are completed.
Apparatuses in accordance with the above features differ from the conventional slit- and filter type imaging spectrometers by utilizing an interferometer as described above, therefore not limiting the collected energy with an aperture or slit or limiting the incoming wavelength with narrow band interference or tunable filters, thereby substantially increasing the total throughput of the system. Thus, these interferometer based spectral imaging systems better utilize all the information available from the incident light of the scene to be analyzed, thereby substantially decreasing the measuring time and/or substantially increasing the signal-to-noise ratio (i.e., sensitivity).
FIG. 1 is a block diagram illustrating the main components of a prior art interferometer based imaging spectrometer disclosed in U.S. Pat. No. 5,539,517.
This imaging spectrometer is constructed highly suitable to implement the method of the present invention as it has high spectral (Ca. 4-16 nm depending on wavelength) and spatial (Ca. 30/M .mu.m where M is the effective microscope or fore optics magnification) resolutions.
Thus, the prior art imaging spectrometer of FIG. 1 includes: a collection optical system, generally designated 20; a one-dimensional scanner, as indicated by block 22; an optical path difference (OPD) generator or interferometer, as indicated by block 24; a one-dimensional preferably two-dimensional detector array, as indicated by block 26; and a signal processor and display, as indicated by block 28.
A critical element in system 20 is the OPD generator or interferometer 24, which outputs modulated light corresponding to a predetermined set of linear combinations of the spectral intensity of the light emitted from each pixel of the scene to be analyzed. The output of the interferometer is focused onto the detector array 26. Thus, all the required optical phase differences are scanned simultaneously for all the pixels of the field of view, in order to obtain all the information required to reconstruct the spectrum. The spectra of all the pixels in the scene are thus collected simultaneously with the imaging information, thereby permitting analysis of the image in a real-time manner.
The apparatuses according to U.S. Pat. No. 5,539,517 may be practiced in a large variety of configurations. Specifically, the interferometer used may be combined with other mirrors as described in the relevant Figures of U.S. Pat. No. 5,539,517.
Thus, according to U.S. Pat. No. 5,539,517, alternative types of interferometers may be employed. These include but are not limited to (i) a moving type interferometer, in which the OPD is varied to modulate the light, namely, a Fabry-Perot interferometer with scanned thickness; (ii) a Michelson type interferometer, which includes a beamsplitter receiving the beam from an optical collection system and a scanner, and splitting the beam into two paths; (iii) a Sagnac interferometer optionally combined with other optical means, in which interferometer the OPD varies with the angle of incidence of the incoming radiation, such as the four-mirror plus beamsplitter interferometer as further described in the cited U.S. patent (see for example FIG. 14 there).
FIG. 2 illustrates an imaging spectrometer constructed in accordance with U.S. Pat. No. 5,539,517 utilizing an interferometer in which the OPD varies with the angle of incidence of the incoming radiation. A beam entering the interferometer at a small angle to the optical axis undergoes an OPD which varies substantially linearly with this angle.
In the interferometer of FIG. 2, all the radiation from source 30 in all the pixels, after being collimated by an optical collection system 31, is scanned by a mechanical scanner 32. The light is then passed through a beamsplitter 33 to a first reflector 34 and then to a second reflector 35, which reflects the light back through the beamsplitter 33 and then through a focusing lens 36 to an array of detectors 37 (e.g., a CCD). This beam interferes with the beam which is reflected by beamsplitter 33, then by second reflector 35, and finally by first reflector 34.
At the end of one scan, every pixel has been measured through all the OPD's, and therefore the spectrum of each pixel of the scene can be reconstructed by Fourier transformation. A beam parallel to the optical axis is compensated, and a beam at an angle (.theta.) to the optical axis undergoes an OPD which is a function of the thickness of the beamsplitter 33, its index of refraction, and the angle .theta.. The OPD is proportional to .theta. for small angles. By applying the appropriate inversion, and by careful book-keeping, the spectrum of every pixel is calculated.
In the configuration of FIG. 2 the ray which is incident on the beamsplitter at an angle .beta. (.beta.=45.degree. in FIG. 2) goes through the interferometer with an OPD=0, whereas a ray which is incident at a general angle .beta.-.theta. undergoes an OPD given by Equation 1: EQU OPD(.beta.,.theta.,t,n)=t[(n.sup.2 -sin.sup.2 (.beta.+.theta.)).sup.0.5 -(n.sup.2 -sin.sup.2 (.beta.-.theta.)).sup.0.5 +2 sin .beta. sin .theta.](1)
where .beta. is the angle of incidence of the ray on the beamsplitter; .theta. is the angular distance of a ray from the optical axis or interferometer rotation angle with respect to the central position; t is the thickness of the beamsplitter; and n is the index of refraction of the beamsplitter.
It follows from Equation 1 that by scanning both positive and negative angles with respect to a central position, one can get a double-sided interferogram for every pixel, which helps eliminate phase errors giving more accurate results in the Fourier transform calculation.
The scanning amplitude determines the maximum OPD reached, which is related to the spectral resolution of the measurement. The size of the angular steps determines the OPD step which is, in turn, dictated by the shortest wavelength to which the system is sensitive. In fact, according to the sampling theorem [see, Chamberlain (1979) The principles of interferometric spectroscopy, John Wiley and Sons, pp. 53-55], this OPD step must be smaller than half the shortest wavelength to which the system is sensitive.
Another parameter which should be taken into account is the finite size of a detector element in the matrix. Through the focusing optics, the element subtends a finite OPD in the interferometer which has the effect of convolving the interferogram with a rectangular function. This brings about, as a consequence, a reduction of system sensitivity at short wavelengths, which drops to zero for wavelengths equal to or below the OPD subtended by the element. For this reason, one must ensure that the modulation transfer function (MTF) condition is satisfied, i.e., that the OPD subtended by a detector element in the interferometer must be smaller than the shortest wavelength at which the instrument is sensitive.
Thus, imaging spectrometers constructed in accordance with the invention disclosed in U.S. Pat. No. 5,539,517 do not merely measure the intensity of light coming from every pixel in the field of view, but also measure the spectrum of each pixel in a predefined wavelength range. They also better utilize all the radiation emitted by each pixel in the field of view at any given time, and therefore permit, as explained above, a significant decrease in the frame time and/or a significant increase in the sensitivity of the spectrometer. Such imaging spectrometers may include various types of interferometers and optical collection and focusing systems, and may therefore be used in a wide variety of applications. including medical diagnostic and therapy and biological research applications, as well as remote sensing for geological and agricultural investigations, and the like.
An imaging spectrometer in accordance with the invention disclosed in U.S. Pat. No. 5,539,517 was developed by Applied Spectral Imaging Ltd., Industrial Park, Migdal Haemek, Israel and is distributed under the name SpectraCube.TM..
The SpectraCube.TM. system optically connected to a variety of optical devices is used to implement the method of the present invention. The SpectraCube.TM. system has the following characteristics, listed hereinbelow in Table 1:
TABLE 1 ______________________________________ Character Performance ______________________________________ Spatial resolution: 30/M .mu.m (M = effective microscope or fore optics magnification) Field of View: 15/M millimeters Sensitivity: 20 milliLux (for 100 msec integration time, increases for longer integration times linearly with .sqroot.T) Spectral range: 400-1000 nm Spectral resolution: 4 nm at 400 nm (16 nm at 800 nm) Acquisition time: 5-50 sec, typical 25 sec FFT processing time: 20-180 sec, typical 60 sec ______________________________________
However, since an interferometer based spectral imager, in order to perform a measurement, must collect several frames of an examined object in a period of time that varies from Ca. 5 to 60 seconds, a considerably longer period of time as compared with a camera or video camera snapshot, spectral imaging of moving objects results in blurring of the image of the object and in disrupting the algorithm used to calculate the spectrum of each pixel thereof.
Indeed, while using the device disclosed in U.S. Pat. No. 5,539,517 one should ensure that the examined object is substantially stationary for best results. This is indeed the case in many applications, such as when spectral imaging is used for color karyotyping and color banding of chromosomes as disclosed in Schroeck et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273:494-497. However, in other applications spectral imaging of a moving object is required. This is the case for example when the examined object is an organ of a living creature (e.g., a human eye or a specific region or tissue thereof).
Spectral images of living organs and tissues can provide important information concerning the chemical makeup of the organ or tissues and thereby provide information concerning for example their metabolic functioning. This is the case since spectroscopy is capable of characterizing objects based on their spectral signatures of chemical constituents, as the physical basis of spectroscopy is the interaction of light with matter.
For example various body substances such as but not limited to hemoglobin, cytochromes, flavins, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and the like may be in either reduced or oxidized forms, each of the forms of each of these substances is characterized by an identifying unique spectrum. Since the oxidation level (i.e., the ratio of the oxidized and reduced forms) of such substances is in many cases correlated to the amount of oxygen arriving at the organ and the level of metabolism, and since changes in the oxidation level are in some cases indicative of a pathological condition, determination of the oxidation level of such substances has been employed using point spectroscopy to determine and estimate the severity of such pathological conditions. One example in which a point spectrophotometer was used for noninvasive measurements of the ocular fundus is described by Delori (1994) Applied Optics 33:7439-7452. However, point spectroscopy is limited as it provides spectral information which is not correlated with spatial information, the way spectral imaging does.
Nevertheless, any attempt to measure a spectral image of a living organ, which organ is not motionless, will result in artifacts and a distorted or particularly noisy spectral image data. If such an image is acquired using filter or grating based spectral imagers, a spatial image registration procedure will be required for best results. Nevertheless, these spectral imagers suffer the above listed limitations. On the other hand, should such an image be acquired by an interferometer based spectral imager which have numerous advantages over other spectral imaging systems, not only spatial registration but also spectral correction will be required.
Thus, the object of the present invention is to provide an interferometer based spectral imaging method by which a spectral image can be measured also in the case where the measured object is not stationary. The modifications the method requires for operation, as compared with the prior art described above, are mostly in the mathematical algorithms used to process the data collected from the moving object, and in some cases also in the way the device is aligned with respect to the examined moving object, which modifications provide both spatial registration and spectral correction.